Toxoplasma gondii is an obligate intracellular parasite which can infect most warm-blooded vertebrates. In humans, it has been recognized as a major cause of severe congenital disease and a common cause of infection in immunocompromised hosts. Recently, the parasite has received increased attention as an important opportunistic pathogen affecting up to 25% of AIDS patients (Kasper, 1992a). In the laboratory, T. gondii is relatively easy to handle and maintain and consequently has become an important model for the study of how obligate intracellular parasites function. To date, however, such studies have been hampered by the absence of a method for introducing DNA into the parasites.
Although transfection and stable transformation have been achieved for a range of trypanosomatids (Bellofatto, et al., 1989; Laban, et al., 1989, 1990; Lee, et al., 1990; Coburn, et al., 1991; Eid, et al., 1991; Lebowitz, et al., 1990; and Tobin, et al., 1991), such methodologies have not been reported for any of the obligate intracellular parasites, most notably members of the phylum Apicomplexa.
Efforts to stably transform these parasites have been complicated by their inability to replicate outside host cells. Neomycin and hygromycin, drugs commonly used for selection of stable transformants in other systems, including protozoan parasites from the kinetoplastida order (Laban, et al., 1989; Lee, et al., 1990; Coburn, et al., 1991), kill host cells as efficiently as parasites.